This invention relates to a novel method for inhibiting tissue ischemia and reperfusion injury and, more particularly, to a method of reducing the extent of tissue injury in the clinical setting of crush injury, amputation, organ transplantation, cerebral vascular diseases (e.g. stroke), ischemic heart diseases (e.g. myocardial infarction), peripheral vascular diseases, and similar such injuries.
Reperfusion injury refers to the cellular changes and tissue damage seen after a period of total ischemia followed by reperfusion. Extremity replantation, organ transplantation, free flap tissue reconstruction and even myocardial infarction and stroke are all clinical examples of interval tissue ischemia which can lead to tissue loss due to reperfusion injury after blood flow is reestablished. Tissue reperfusion injury, seen in its full clinical extent as the no-reflow phenomenon, appears as an inflammatory response to reperfusion resulting in the ultimate death of the tissue.
The tissue injury that occurs after a period of total ischemia and subsequent revascularization (reperfusion injury) has several proposed coincident mechanisms that often lead to either partial or complete tissue necrosis. The extent of injury primarily depends upon the length of time and degree of ischemia as well as the type of tissues affected. The pathology of the observed tissue injury includes vascular and cellular responses [Peacock, in Wound Repair, (E. E. Peacock, ed.) Saunders, Philadelphia, p. 1 (1984); and Davis and Allison, in Handbook of Experimental Pharmacology, (J. R. Vane and S. H. Ferreira, eds.) Springer, N.Y., p. 267 (1978)]:
a) The vascular response involves an increased vessel wall permeability to plasma and macromolecules, which is responsible for the edema formation; PA1 b) The cellular response is characterized by the appearance of neutrophils; PA1 c) Lysosomal enzymes and other mediators, such as eicosanoides, released from these cells after specific stimuli or cell death may contribute to tissue damage and necrosis; and PA1 d) Intravascular thrombosis. PA1 the formation of fibrin; PA1 the aggregation of platelets [Berndt and Phillips, in Platelets in Biology and Patholoqy, (E. D. Gordon, ed.) Elsevier/North Holland Biomedical Press, Amsterdam, 1981, pp. 43-74]; PA1 the adherence of neutrophils to vessel wall by an endothelium-dependent mechanism [Zimmerman et al., Ann. N.Y. Acad. Sci. 485, 349-368 (1986)]; PA1 the chemotaxis of monocytes [Bar-Shavit et al., J. Cell Biol. 96, 282-285 (1983)]; PA1 the mitogenic proliferation of lymphocytes, fibroblasts, and vascular smooth muscle cells [Carney et al., Sem. Thromb. Hemost. 18, 91-103 (1992)]; and PA1 the modulation of vascular functions [Blusa, Sem. Thromb. Hemost 18, 296-304 (1992)].
In the clinical setting, while the tissue injury caused by the period of anoxia may be fixed and irreversible, further tissue injury by the circulating blood cells, intravascular thrombosis and tissue edema may be preventable. A multitude of agents have been studied and reported heretofore with varying degrees of limited success. No one agent has been demonstrated to have superior effects such that it has moved into common clinical usage.
The mechanism of reperfusion injury is highly complex and remains only partially understood. It is believed that xanthine dehydrogenase is converted to xanthine oxidases within the endothelial cell during ischemia, permitting the production of superoxide (O.sub.2 --) which causes the initial endothelial cell injury. Secondarily, the superoxide may activate neutrophils which act as an amplifier of the initial injury [Sussman and Bulkley, Methods Enzymol. 186, 711-723 (1990)]. In response to endothelial/vascular injury, multiple cellular and humoral activations occur that mediate a host of hemostatic and inflammatory processes. During the hemostatic process, a number of products are generated which are proinflammatory. For example, the product of the coagulation cascade, thrombin, has been shown to activate cells involved in the inflammatory process, such as endothelial cells, platelets, neutrophils, monocytes and smooth muscle cells [M. A. Shuman, Ann. NY Acad. Sci. 485, 228-239 (1985); and Carney et al., Sem. Thromb. Hemost. 18, 91-103 (1992)]. Further evidence that coagulation is closely linked to inflammation has recently been reviewed by Esmon et al., Thromb. Haemost. 66 160-165, (1991). The expression of tissue factor on the damaged endothelium and activated monocyte/macrophages is thought to initiate the coagulation cascade [Nemerson, Sem. Hematol. 29, 170-176 (1992); and Rapaport and Rao, Arteriosclerosis Thromb. 12, 1111-1121 (1992)] and this expression has been shown to be greatly enhanced under inflammatory conditions [Edwards and Rickles, Sem. Hematol. 29, 202-212 (1992)]. These considerations suggest that connection between coagulation and inflammation occurs during vascular injury, and inhibition of the tissue factor activity in the injured vessels may lead to modulation of inflammatory response.
A large body of work heretofore has focused especially on the role of leukocytes (neutrophils in particular) and oxygen-derived free radicals in ischemia injury. Using a cat small intestine model, it was shown that anti-neutrophil serum or monoclonal antibody that inhibits neutrophil adherence to endothelial cells prevented reperfusion-induced capillary leak [Hernandez et al., Am. J. Physiol. 253, H699-703 (1987)]. In many other models it was shown that scavengers of oxygen metabolites (e.g. superoxide dismutase, catalase, dimethyl sulfoxide), chelators of iron (e.g. desferrioxamine, transferrin), and inhibitors of xanthine oxidase (e.g. allopurinol, pterin aidehyde, tungsten feeding) provided protection from ischemia/reperfusion injury [Sussman and Bulkley, Methods Enzymol. 186, 711-723 (1990)]. However, oxygen-free radical scavengers have not always been reported to be beneficial in all systems studied and they do not prevent all of the injury patterns noted [Winchell and Halasz, Transplantation 48, 393-396 (1989)]. Steroids and other synthetic compounds that exert their anti-inflammatory action by reducing the availability of arachidonic acid for conversion to prostaglandins and leukotrienes were found to inhibit the progression of necrosis in an ischemic rabbit ear model [Hayden et al., Prostaglandins 33, 63-73 (1987)]. Use of anti-thrombotic agents has also reportedly shown improved results after reperfusion. For example, streptokinase and urokinase have been shown to improve the rat epigastric flap [Jacobs et al., Plast. Reconstr. Surg. 68,737 (1981)] and replanted limbs [Zdeblick et al., J. Bone Joint Surg. 69A, 442 (1987)], respectively. However, anticoagulants alone may not provide maximal tissue protection, since reperfusion injury was found to occur in defibrinated animals [Simpson et al., J. Pharmacol. Exp. Ther. 256, 780-6 (1991)]. Furthermore, it is interesting to note that epigastric flaps survived better by the combined use of anticoagulants (heparin and urokinase) with superoxide dismutase and catalase [Maeda et al., J. Reconstr. Microsurg. 7, 233-243 (1991)]. All these results taken together suggest that ischemia/reperfusion injury involves a complex interplay of many biochemical, humoral and cellular pathways, all of which may contribute to the eventual tissue injury. Compounds that inhibit various points of the pathways may lead to different degrees of modulation of the complex cascade of events that lead to tissue injury and necrosis.
Other recently proposed drug treatments for ameliorating tissue damage from ischemia and reperfusion include the use of a non-anticoagulant heparin as described in PCT WO 94/08595, published Apr. 28, 1994, and detoxified endotoxins monophosphoryl lipid A or 3-deacylated monophospholipid A as disclosed in U.S. Pat. No. 5,286,718, dated Feb. 15, 1994.
It is known that plasma contains a multivalent Kunitz-type inhibitor of coagulation, referred to herein as tissue factor pathway inhibitor (TFPI). This name has been accepted by the International Society on Thrombosis and Hemostasis, Jun. 30, 1991, Amsterdam. TFPI was previously known as lipoprotein-associated coagulation inhibitor (LACI). TFPI was first purified from a human hepatoma cell, Hep G2, as described by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987), and subsequently from human plasma as reported by Novotny et al., J. Biol. Chem. 264, 18832-18837 (1989); and Chang liver and S. K. hepatoma cells as disclosed by Wun et al., J. Biol. Chem. 265, 16096-16101 (1990). TFPI cDNA have been isolated from placental and endothelial cDNA libraries as described by Wun et al., J. Biol. Chem. 263, 6001-6004 (1988); and Girard et al., Thromb. Res. 55, 37-50 (1989). The primary amino acid sequence of TFPI, deduced from the cDNA sequence, shows that TFPI contains a highly negatively charged amino-terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. The first Kunitz domain of TFPI is needed for the inhibition of the factor VII.sub.a /tissue factor complex, and the second Kunitz domain of TFPI is responsible for the inhibition of factor X.sub.a according to Girard et al., Nature 328, 518-520 (1989), while the function of the third Kunitz domain remains unknown. See also U.S. Pat. No. 5,106,833. TFPI is believed to function in vivo to limit the initiation of coagulation by forming an inert, quaternary factor X.sub.a : TFPI: factor VII.sub.a : tissue factor complex. Further background information on TFPI can be had by reference to the recent reviews by Rapaport, Blood 73, 359-365 (1989); and Broze et al., Biochemistry 29, 7539-7546 (1990).
Recombinant TFPI has been expressed as a glycosylated protein using mammalian cell hosts including mouse C127 cells as disclosed by Day et al., Blood 76, 1538-1545 (1990), baby hamster kidney cells as reported by Pedersen et al., J. Biol. Chem. 263, 16786-16793 (1990), Chinese hamster ovary cells and human SK hepatoma cells. The C127 TFPI has been used in animal studies and was shown to be effective in the inhibition of tissue factor-induced intravascular coagulation in rabbits according to Day et al., supra, and in the prevention of arterial reocclusion after thrombolysis in dogs as described by Haskel et al., Circulation 84, 821-827 (1991).
Recombinant TFPI also has been expressed as a non-glycosylated protein using E. coli host cells and obtaining a highly active TFPI by in vitro folding of the protein as described in U.S. Pat. No. 5,212,091, the disclosure of which is incorporated by reference herein. See also Wun et al., Thromb. Hemostas. 68, 54-59 (1992).
The cloning of the TFPI cDNA which encodes the 276-amino acid residue protein of TFPI is further described in Wun et al., U. S. Pat. No. 4,966,852, the disclosure of which is incorporated by reference herein.
Recently, TFPI obtained through recombinant DNA clones expressed in E. coli as disclosed in U.S. Pat. No. 5,212,091, has been described as useful for reducing the thrombo-genicity of microvascular anastomoses. See U.S. Pat. No. 5,276,015, the disclosure of which is incorporated herein by reference.
The use of TFPI for treatment of sepsis or septic shock and sepsis-associated disorders is described in recently published patent applications PCT WO 93/24143 and PCT WO 93/25230.